Description: (Verbatim from the Applicant's Abstract) The long-term goals of the proposed work are to decipher the unique cellular functions of enzymes, both catalytic and "moonlighting" functions. Fructose-1,6-bisphosphate aldolase isozymes A, B, and C are crucial to metabolism in the cell, have proven to be defectivein lethal genetic diseases, and are drug targets for parasitic disease. The human isozymes share as much as 81 percent sequence identity, yet the catalytic efficiencies toward fructose-1,6-bisphosphate and fructose-1-phosphate differ by as much as two orders of magnitude between isozymes. Furthermore, aldolase isozymes have been demonstrated to bind to several important intracellular binding targets, such as F-actin and the calcium-binding S100 proteins. Evolutionary comparisons have revealed isozyme-specific residues (ISRs) and this proposal will determine which of these ISRs confer isozyme-specific functions, both in terms of their differential catalytic function and activity in protein-protein interactions. Three models will be tested: 1) binding to cellular target proteins modulates aldolase catalytic activity in an isozyme-specific manner, 2) the binding of aldolase to cellular target proteins is distinct from catalytic function and represents a "moonlighting" function of aldolase in modulating the activity of other proteins, and 3) the binding of aldolase to cellular target proteins locates aldolase (and its activities) in the cell. These models will be tested by asking: 1) can the different binding modes for substrates and enzymatic intermediates among the three aldolase isozymes be correlated to the functional differences reflected in the rate-limiting steps? 2) Do the patches of ISRs on the protein surface correlate with these functions or with moonlighting functions? This will be tested by measuring the binding interactions of aldolase isozymes to S100A1 and F-actin, and testing the same with chimeras wherein the ISR patches of one isozyme have been swapped with another isozyme. 3) What is the structure of one of the two patches of ISRs that are localized in the carboxyl-terminal region of aldolase? The proposed research will determine the complete structures of each aldolase isozyme (A, B, and C) with and without carboxyl-terminus by a combination of TROSY NMR and segmental isotopic labeling using intein-mediated protein ligation, and the substrate- specific kinetic differences and their respective rate-limiting steps by presteady-state kinetics. This work is important for understanding the roots of isozyme specificity and the interplay between catalytic and cellular functions of enzymes so critical to the field of functional genomics.